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Volumn 283, Issue 5407, 1999, Pages 1538-1538

Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1

Author keywords

[No Author keywords available]

Indexed keywords

GENE PRODUCT; MEMBRANE PROTEIN; PROTEIN GLUTAMINE GAMMA GLUTAMYLTRANSFERASE; FUNGAL PROTEIN; GUANINE NUCLEOTIDE BINDING PROTEIN; GUANOSINE TRIPHOSPHATASE; HWP1 PROTEIN, CANDIDA ALBICANS; RECOMBINANT PROTEIN; TRANSGLUTAMINASE 2;

EID: 0345391036     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5407.1538     Document Type: Article
Times cited : (506)

References (33)
  • 9
    • 0344148903 scopus 로고    scopus 로고
    • note
    • 2-terminus of the mature protein through the proline- and glutamine-rich-repeats [amino acids 40 to 197 encoded by HWP1 cDNA (2)], was purified from culture media of Pichia pastoris by anion-exchange chromatography (Mono Q, Bio-Rad), with a salt gradient (0 to 1 M NaCl) in column buffer [50 mM tris-HCl (pH 8), 2 mM mercaptoethanol, 1 mM EDTA].
  • 15
    • 0023922945 scopus 로고
    • L. Lorand, O. M. Lockridge, L. K. Campbell, R. Myhrman, J. Bruner-Lorand, Anal. Biochem. 44, 221 (1971); P. T. Velasco, F. Karush, L. Lorand, Biochem. Biophys. Res. Commun. 152, 505 (1988); L. Lorand, L. K. Campbell-Wilkes, L. Cooperstein, Anal. Biochem. 50, 623 (1972).
    • (1988) Biochem. Biophys. Res. Commun. , vol.152 , pp. 505
    • Velasco, P.T.1    Karush, F.2    Lorand, L.3
  • 16
    • 0015463837 scopus 로고
    • L. Lorand, O. M. Lockridge, L. K. Campbell, R. Myhrman, J. Bruner-Lorand, Anal. Biochem. 44, 221 (1971); P. T. Velasco, F. Karush, L. Lorand, Biochem. Biophys. Res. Commun. 152, 505 (1988); L. Lorand, L. K. Campbell-Wilkes, L. Cooperstein, Anal. Biochem. 50, 623 (1972).
    • (1972) Anal. Biochem. , vol.50 , pp. 623
    • Lorand, L.1    Campbell-Wilkes, L.K.2    Cooperstein, L.3
  • 18
    • 0027192868 scopus 로고
    • + homozygous hwp1/hwp1 strain, CAH7-1A1, with Hind III-digested pGBHWP1 and p24enura (20) digested with Xba I-Xho I, resulting in an enolase gene disrupted with URA3. Generation times of CAH7, CAH7-1A, and CAHR3 were 2.52, 2.57, and 2.57 hours, respectively, whereas that of SC5314 was 2.00 hours in YNB at 27°C.
    • (1993) Genetics , vol.134 , pp. 717
    • Fonzi, W.A.1    Irwin, M.Y.2
  • 19
    • 0345011399 scopus 로고    scopus 로고
    • note
    • Hwp1 was assessed with monospecific antiserum to rHwp1 in indirect immunofluorescence assays (2).
  • 20
    • 0345442497 scopus 로고    scopus 로고
    • note
    • 2, 1 mM EDTA, 1 mM dithiothreitol (DTT)], at a germ tube/BEC ratio of 100:1 and incubated for 1 hour at 37°C, followed by reaction termination with 100 mM ECTA (75 μl). For overall adhesion, radioactivity of BEC fractions was determined by scintillation counting after centrifugation on 50% Percoll gradients (27), and the number of germ tubes per BEC was determined with the specific activity of each strain. For stabilized adhesion assays, reaction mixtures were heated to 100°C for 2 min in phosphate-buffered saline (PBS) containing 1% SDS before loading on Percoll gradients. Monodansylcadaverine (5 mM) or iodoacetamide (10 mM) was used to inhibit TGase during the 1-hour incubation at 37°C. Subtracted background counts, determined from gradients lacking BECs, were less than 6% of counts for BEC-associated counts. Visual counting confirmed results with radiolabeled cells.
  • 22
    • 0345442496 scopus 로고    scopus 로고
    • note
    • 5 BECs in 300 μl of reaction buffer 3 under stabilized adhesion assay conditions (17). BECs were washed in 500 μl of PBS, suspended in 50 μl of sample buffer (23), boiled for 5 min, vortexed, and centrifuged. Supernatants containing unbound proteins were discarded and BECs were suspended in PBS (50 μl) and transferred to vials with scintillation fluid to determine BEC-associated dpm.
  • 28
    • 0344148895 scopus 로고    scopus 로고
    • note
    • 2,1 mM DTT, 1 mM EDTA] for 30 min at 37°C (total volume, 25 μl). Radioactivity bound to acceptor proteins was assessed by scintillation counting after trichloroacetic acid precipitation. Background counts from reactions without acceptor proteins were subtracted from total counts (13).
  • 29
    • 0345011398 scopus 로고    scopus 로고
    • note
    • Reactions (25) were incubated for 4 hours and quenched with cold putrescine (100 mM) for 1 hour before boiling in Laemmli sample buffer (23) for 5 min.
  • 32
    • 0345442495 scopus 로고    scopus 로고
    • note
    • Organisms were grown for 18 hours in phytonepeptone broth supplemented with 0.1% glucose at 25°C with shaking at 100 rpm.
  • 33
    • 0345011394 scopus 로고    scopus 로고
    • note
    • Animal studies were conducted in accordance with the NIH guidelines for the care and use of laboratory animals. We thank A. Jackson for assistance with preparation of TGase2, B. Taylor for assistance with the animal experiments, and C. M. Allen for histological assessments of infected mouse tissues. Support for this research was provided from grant 1 RO1 DE11375-05A2 from the National Institute of Dental and Craniofacial Research (P.S.) and AI32556 from the National Institute for Allergy and Infectious Diseases (P.L.F.).


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